Effects Of Leukamenin E, An Ent-kaurane Diterpenoid, On Differention Induction And Cytoskeleton Of Hela Cells

Cancer is the major disease imperiling human health badly. The Radiotherapy andSurgical therapy are main cancer tretment. Inducing tumor cell differentiation becomes thenew cancer treatment. To compared with traditional cancer treatment, this treatment hasadvantages of small dosage and low toxic effect.Isodon plants widely distributed in China and variety. The results show that the contentof diterpenoids was very rich and major active ingredient in Isodon plants. It is reported thatmost of them to be used anti-tumor. So far, about Leukamenin E could induce Hela celldifferentiation, and there are few literature report the effect of Leukamenin E and WangzaozinA on the Hela cell cytoskeleton.In this paper, ent-kaurane diterpenoids Leukamenin E(from(Isodonexcisoides(Sun exC.H.Hu)C.Y_Wu et H.w.Li) in Gansu Province) was used as subjects compound to text theeffects on Hela cell growth with trypan blue exclusion method. In addtion, the effect ofcompounds for cell cycle were detected. At same time, inducing Hela cell differentiation wasstudied by Tablet colony formation, demermination of alkaline phosphatase and Giemsastaining. At last, we observe the change of cytoskeleton with different concentration ofLeukamenin E. The result as follows:(1)The results of trypan blue staining was demonstrated that Leukamenin E has obviousinhibition on the growth of cells a time and dose dependent manners. At the same time anddifferent concentrations, as the concentration increases, the growth inhibition was enhanced;at the same concentrations and different time conditions, the higher the concentration, theslower growth rate.(2)Tablet colony forming experiment was demonstrated that Leukamenin E has obviousinhibition on colony forming ability of Hela cells. And there is a significant differencebetween the different concentrations.(3)Inverted microscope observd morphological changes: control group of cellmorphology was normal, no significant changes, arranged densely, only to see a few floatingcells. as the concentration increases, the number of cells was reduced and showed a time-andconcentration-dependent, cell arrangement sparse, irregular shape changed irregularly,irregular protrusions extending long, there were many floating mulberry-like cells in culturemedium. (4)Giemsa staining is demonstrated that Leukamenin E has a significant influence onHela cell nucleus. the shape of cell nucleus is similar to kidney. Cell volume is increasedcompared with control and the ratio nucleoplasm was decreased. With the increase of drugconcentration and the extension of time, this phenomenon was more obvious. At same time,small vacuoles was appeared in the cytoplasm.(5)Flow cytometry displayed that the distribution of cell cycle could be changeed withthe treatment of Leukamenin E. It could induce strong G1phase in time-dependent, cellcontent of S phase had slightly decline.(6)Rcactive oxygen detection displayed that the content of ROS in cell was increasedwith the treatment of Leukamenin E, we speculated that ROS would start the celldifferentiation.(7) The changes of alkaline phosphatase activity: After Hela cells were treated withdifferent concentrations of Leukamenin E,alkaline phosphatase activity was significantlyincreased. Compared with control group, significant differences were exist.(8)Scratch cell migration results displayed that Leukamenin E had strong inhibitioneffect to Hela cell migration. With the increase of the concentration, Inhibition effect wasmore obvious, and at the highest concentration, cell migration was completely inhibited.(9)We observed the change of F-actin by indirect immunofluorence staining,Leukamenin E could destroy microfilaments with the increase of the concentration. Thecontent of microfilaments in control was rich and parallel stress fibers arranged clutter. Atlower concentration, stress fibers arranged neat and along the longitudinal axis. With theincrease of the concentration, the distribution of stress fibers become non-uniform anddisappeared at the highest concentration, and the content were significantly reduced.(10)Tubulin are showing irregular-shaped filament structure or network structure withdifferent concentration. Microtubules evenly distributed in control cells, after-treatment, theoverall trend was gathered to the nucleus, and with extension of time and increasedconcentration, aggregation phenomenon was more obvious. One strongly fluorescencemacular area is observed perinuclear. Microtubules from nuclear week, firing around.(11)Keratin Ifs are showing irregular-shaped filament structure or network structure withdifferent concentration. Keratin Ifs evenly distributed in control cells. At low concentrations,the structure of Keratin Ifs had no change when comparaed with control. With the increase of the concentration, the overall trend was gathered to the nucleus, and with extension of timeand increased concentration, aggregation phenomenon was more obvious. One stronglyfluorescence macular area is observed perinuclear. Keratin Ifs from nuclear week, firingaround, significantly occurred rearrangement.(12)Flow cytometry displayed that the content of three kinds Cytoskeleton were changed.the content of Microfilaments was decreased with the increase of the concentration, while thecontent of Microtubules and Keratin Ifs were increased with increase of the concentration.